Reverse DNA gyrases are enzymes found in thermophilic bacteria, whose action introduces positive supercoiling into circular DNA at the expense of ATP hydrolysis. Previously described enzymes of this class have a single polypeptide chain. We have now found a reverse gyrase with an unusual structure in the hyperthermophile Methanopyrus kandleri. This enzyme has two subunits, one having the ATPase activity while the other carries out the DNA breakage-reunion step. This functional partition is similar to that of the eubacterial DNA gyrases. Further studies of E. coli DNA gyrase have shown that the coupling between supercoiling and nucleotide triphosphate consumption depends on the initial supercoiling of the DNA. Tight coupling can be obtained if the DNA is initially positively supercoiled.